KR101448121B1 - Method for identifying duck using multiplex PCR - Google Patents

Abstract

The present invention relates to a composition for identifying ducks comprising at least one primer pair selected from the group consisting of primer pairs consisting of the nucleotide sequences represented by SEQ ID NOS: 1 to 24, and a method for identifying ducks using the same. The composition for identifying ducks according to the present invention can selectively detect alleles and genotypes by selectively amplifying a supersatellite locus having a discriminating power in the duck group, have.

Description

Method for identifying ducks using multiplex PCR [

The present invention relates to a composition for identifying ducks comprising at least one primer pair selected from the group consisting of primer pairs consisting of the nucleotide sequences represented by SEQ ID NOS: 1 to 24, and a method for identifying ducks using the same.

Recognizing the value of conservation and utilization of native livestock as a genetic resource in the world (Notter (1999) J. Anim . Sci . 77: 61-69), breed formation, genetic characteristics, genetic diversity, And the relationship between the two. Recently, various genetic analysis methods based on DNA polymorphism have been developed and identification has been made between varieties and individuals (Peelman et al., 1998. Evaluation of the genetic variability of bovine microsatellite markers in four Belgian cattle breeds. Anim., Genet., 29: 161-167).

Microsatellite (MS) is a repetitive DNA group of about 2 to 6 nucleotides, which is a non-coding DNA sequence that is uniformly distributed within the genome and exhibits a very high polymorphism (Zajc I and Sampson J (1999) J Hered 90: 104-107). Variability among individuals is recognized according to the number of repeats in a particular locus (Koreth J, O'Leary JJand McGee JO (1996) J Pathol 178: 239-248.), When the polymerase chain reaction (PCR) is performed using a primer designed in the region, the polymorphism of the PCR product length is observed, and it becomes possible to detect the DNA polymorphism. In addition, the supersatellite, like other genes, is transmitted to the offspring according to the Mendel's law of genetics and can be amplified by PCR and co-dominant when electrophoresed. In particular, since the supersatellite is small in size, it can be amplified from two to eight primers simultaneously (Chamberlain JS et al., (1988) Nucleic Acids Res 16: 11141-11156) They can reduce costs and easily analyze their allelic forms.

This supersatellite analysis has been widely carried out all over the world, and it has been widely used in domestic as well as academic researches, beef history management, development of cattle and imported meat discrimination method.

Unlike the traditional popular meat, beef and pork, ducks have been recognized as a healthy meats since the mid-2000s, and have been widely recognized as a health food. As the popularity of these ducks grows, there is a growing need for standards to assess the quality of duck meat, such as beef and pork. As a result, the Livestock Products Quality Assessment Service has been conducting duck meat grading since November 21, 2011. However, this is a class that judges and assigns the appearance, the fattening condition, the fat adhesion, the fleece, the feather, the freshness, and the trauma collectively, and is not applicable to the quality control according to the individual identification of the duck. Thus, there is a need for a method of rapidly identifying individuals of a duck, such as beef or pork.

Therefore, it is necessary to study the method of effectively identifying ducks using the ultra-satellites locus, because it is advantageous to effectively control the quality of the ducks if the ducks can be identified while saving time and money.

The present inventors have been studying to develop an effective method for identification of ducks by accurately analyzing the alleles of supersatellator loci and standardizing them so as to select the supersatellite locus having discriminative power in the duck group, To identify ducks, thus completing the present invention.

It is an object of the present invention to provide a composition for identifying ducks comprising at least one primer pair specific to a supersatellite locus allele for identifying ducks, and a method for identifying ducks using the same.

The present invention provides a polypeptide comprising the amino acid sequences of SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: , SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22; And at least one primer pair selected from the group consisting of primer pairs consisting of the nucleotide sequences shown in SEQ ID NOs: 23 and 24, and a kit for identifying ducks comprising the same.

The present invention also relates to a method for producing a nucleic acid comprising the steps of: 1) obtaining a nucleic acid sample from a duck subject to be identified; 2) amplifying the nucleic acid sample of step 1) by multiplex PCR using a primer specific to the supersatellite locus of the duck; 3) electrophoresing the amplified product of step 2); And 4) detecting an allele in the electrophoresis product of step 3) to determine a genotype.

The method of identifying a duck according to the present invention has an excellent effect of promptly and accurately identifying a duck object by selectively amplifying a supersatellite locus having a discriminating power in a duck group and detecting an allele gene and determining a genotype .

FIG. 1 is a diagram showing the result of analyzing allele genotypes by multiplexing 12 loci of super-satellites to identify ducks by multiplex PCR.

The present invention provides a polypeptide comprising the amino acid sequences of SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: , SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22; And at least one primer pair selected from the group consisting of primer pairs consisting of the nucleotide sequences shown in SEQ ID NOs: 23 and 24, and a kit for identifying ducks comprising the same.

The composition for identifying ducks and the kit for identifying ducks comprising the same selectively amplify a supersatellite locus having a discriminating power in a duck group to detect alleles and determine a genotype so that duck objects can be identified quickly and accurately .

The primer is characterized by being capable of amplifying a supersatellite locus having a discriminating power in a duck group, and the supersatellator locus is characterized in that the locus of the supersatellator is selected from the group consisting of CAUD055, CAUD065, CAUD086, CAUD074, CAUD004, AMU006, APH21, AMU060, CAUD120, CAUD083, AMU003 and CAUD130 , And the like.

The number of alleles is at least 7 and the size of the amplified product is 80bp to 300bp. The number of alleles is at least 7 and the size of the amplified product is 80bp to 300bp Is preferably used.

 The primer may be any primer that can replace a part of the base sequence with another base or that can specifically amplify the supersatellite locus even if the position and orientation of some base sequences are changed, can do.

The duck identification kit may be a kit that identifies an amplification product amplified from a nucleic acid sample collected from a duck to be identified using the primer set and identifies the individual through a pattern of the amplification result. The kit may include amplification means capable of amplifying the nucleic acid sample and detection means for detecting the gene marker from the amplified product. The amplification means may be a conventional means for performing a polymerase chain reaction. For example, the amplification means may be a reaction mixture capable of performing a polymerase chain reaction using a polymerase chain reaction (PCR). The reaction mixture may contain reaction buffer, Taq DNA polymerase, dNTP, and MgCl₂. When the PCR reaction is a real-time PCR reaction, fluorescence resonance energy transfer (FRET) and measuring the degree of fluorescence.

The amplification means may further comprise a polymerase chain reaction reactor in addition to the reaction mixture. The polymerase chain reaction may be a conventional polymerase chain reaction, and may be, for example, a real-time PCR, a thermal block PCR, or a micro-PCR However, the present invention is not limited thereto. The detection means for detecting the individual specificity from the amplification product amplified by the amplification means may be a means capable of detecting a common gene marker and preferably the number of DNA bands of the result of electrophoresis of the amplification product or the DNA The number of bands, the size, or the annealing temperature at which the double helix of the amplified DNA is separated.

The present invention also relates to a method for producing a nucleic acid comprising the steps of: 1) obtaining a nucleic acid sample from a duck subject to be identified; 2) amplifying the nucleic acid sample of step 1) by multiplex PCR using a primer specific to the supersatellite locus of the duck; And 4) detecting an allele in the electrophoresis product of step 3) to determine a genotype.

The primers may be at least two selected from the group consisting of CAUD055, CAUD065, CAUD086, CAUD074, CAUD004, AMU006, APH21, AMU060, CAUD120, CAUD083, AMU003 and CAUD130. And SEQ ID NOS: 7 and 8, SEQ ID NOS: 9 and 10, SEQ ID NOS: 11 and 12, SEQ ID NOS: 13 and 14, SEQ ID NOS: 15 and 16, SEQ ID NOS: 17 and 18 , SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22; And at least one primer pair selected from the group consisting of primer pairs consisting of the nucleotide sequences shown in SEQ ID NOs: 23 and 24.

Hereinafter, the duck entity identification method will be described step by step.

The step 1) is a step of obtaining a nucleic acid sample from a duck, and genomic DNA can be isolated from the blood of a duck using a genomic DNA extraction kit.

The nucleic acid sample may be not only the blood of the duck to be identified, but also genomic DNA sampled from the semen of the duck, juicy or duck tissue.

More specifically, the method of extracting the genomic DNA can be carried out by extracting DNA from ordinary blood when the DNA sample is a blood sample. For example, genomic DNA extraction kit such as genomic DNA extraction kit or FlexiGene DNA kit And extracting DNA using a kit. In addition, when the DNA sample is a duck tissue, it can be carried out by extracting genomic DNA from an ordinary animal tissue. For example, after the tissue is pulverized using a homogenizer or the like, And extracting DNA using a genomic DNA extraction kit such as a DNA extraction kit or a FlexiGene DNA kit. When the DNA sample is a semen sample, it can be performed by extracting genomic DNA from a normal animal sperm. For example, after separating sperm from the animal sperm, extracting genomic DNA from the sperm cell And the like.

The step 2) is a step of amplifying the nucleic acid sample by multiplex PCR using a primer specific to a supersatellite locus, and is represented by SEQ ID NOS: 1 to 24, which is a primer specific to a supersatellite locus for analysis of oligomorphism shown in Table 1 Amplified using primer pairs.

In addition, the primer may be labeled with a fluorescent substance, and is preferably labeled only in one of the pair of amplification primers. The fluorescent substance to be labeled may be a fluorescent substance for biological analysis, for example, but not limited to, FAM, VIC, NED, PET, and the like.

The multiplex PCR can be carried out under the usual conditions of polymerase chain reaction, but it is preferable that denaturation is carried out at 95 ° C for 15 minutes, followed by 50 seconds at 95 ° C, 70 seconds at 56 ° C and 70 seconds at 72 ° C 1 cycle. It is then most preferable to perform the PCR at 65 ° C for 30 minutes and then terminate the reaction at 8 ° C.

The step 3) may be performed to electrophore the amplified products in step 2), and to confirm amplification and concentration of each PCR final product for the locus locus analysis. Preferably, the electrophoresis is performed primarily on 3% agarose gel and secondary electrophoresis is performed on an Applied Biosystems 3130xl Genetic Analyzer, which is an automatic base sequence analyzer.

In step 4), the allele is detected in the electrophoresis product of step 3) and the genotype is determined. The determination of the genotype may be accomplished by determining the magnitude of the amplicons of each supersatellite locus and the type of marker and deriving the outcome.

The step of detecting the allele and determining the genotype in the electrophoresis product may be performed by analyzing the result of electrophoresis of the amplification product, and the method may further include the steps of: Or the like. For example, in the case of a sample collected from a duck, based on the fact that the DNA band as a result of the electrophoresis shows a tendency to appear as a single band or a plurality of bands, To identify the entity.

The method for determining the size of the DNA may be a conventional method for checking the size of the DNA, and may be performed by, for example, electrophoresis.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example  1. Duck-specific Super satellites Locus  Selection

Genomic DNA was isolated from the blood of ducks using a genomic DNA extraction kit (Qiagen, USA) to obtain identification samples.

Specific locus of super-satellites for identification of ducks was obtained from ISAG (Applied Biosystems, Inc., Stockmarker International Society for Animal Genetics, http://www.isag.org.uk/ISAG/all/02_PVpanels_LPCGH.doc) for polymorphism analysis of ducks The supersatellite loci were selected as the basis. Table 1 shows the selected 12 supersatellite loci.

Gene GenBank Accession No . Allele No . CAUD055 AY493300 5 CAUD065 AY493310 4 CAUD086 AY493331 4 CAUD074 AY93319 4 CAUD004 AY493249 5 AMU006 AB180491 4 APH21 AJ515896 5 AMU060 AB180542 4 CAUD120 AY587039 7 CAUD083 AY493328 4 AMU003 AB180488 4 CAUD130 AY587049 4

Example  2. Super satellites Locus  Specific Primer  making

The locus specific primers were custom designed by Applied Biosystems (USA), and the fluorescent material was labeled only in one of the pair of amplification primers. 6-FAM (6-carboxylfluorescein), VIC, NED, and PET were used as fluorescent materials. CAUD055, CAUD065, and CAUD086 primers were amplified by FAM, primers for CAUD074, CAUD004 and AMU006 gene amplification were VIC, primers for amplification of APH21, AMU060 and CAUD120 genes were NED, CAUD083, AMU003 and CAUD130 gene amplification Primers were fluorescently labeled with PET.

The supersatellite locus specific primer sequences used in the present invention are shown in Table 2 below.

Example  3. Multiplex PCR Using Super satellites Locus  analysis

Multiplex PCR was carried out to a final volume of 15 μl, 50 ng of template DNA, 2 μl of primer mixture, and dNTP concentration of 0.25 mM. In addition, 0.5 unit of Taq DNA polymerase (Zenchuck Co., Ltd.) and 1.5 μl of 10 × buffer were added and the final volume was adjusted with sterilized tertiary distilled water. The PCR conditions were denaturation at 95 ° C for 15 minutes, followed by 32 cycles of 95 ° C for 50 seconds, 56 ° C for 70 seconds, and 72 ° C for 70 seconds as one cycle. The PCR was then carried out at 65 ° C for 30 minutes and the PCR reaction was terminated at 8 ° C.

For the analysis of supersatellite loci, amplification status and concentration of each PCR final product were confirmed by primary electrophoresis on 3% agarose gel. Then, it was diluted with about 80 times of distilled water for the second electrophoresis in an Applied Biosystems 3130xl Genetic Analyzer (Applied Biosystems, USA), which is an automatic sequencing apparatus. To fractionate the PCR product by size, a mixture of GeneScan (TM) -500 LIZ size standard and Hi-Di formamide (Applied Biosystems, USA) was diluted 1: 9 and subjected to electrophoresis. The allelic ladder for all supersatellites was diluted 1: 9 with a mixture of GeneScan (TM) 500 LIZ Size Standard and Hi-Di Formamide (USA) as well as electrophoresis of PCR final products. Respectively. After electrophoresis, amplification size and marker species of each supersatellite locus were identified using GeneMapper 4.0 (Applied Biosystems, USA) and the results were obtained.

Obs-Ht (polymorphic information content) (PIC) and repeating sequence (polymorphic information index) according to the genome locus of the supersatellite are shown in Table 3 below. The following results were calculated using Cervus version 2.0. The repeat unit means the number of times the nucleotide sequence within each parenthesis is repeated.

Locus Allele No. Obs-Ht PIC Repeat CAUD055 5 0.54 0.53 (AC) 17 CAUD065 4 0.27 0.28 (CA) 17 CAUD086 4 0.71 0.64 (AC) 10 CAUD074 4 0.6 0.49 (CA) 12 CAUD004 5 0.59 0.56 (AC) 20 AMU006 4 0.44 0.42 (CA) 11 APH21 5 0.65 0.57 (CA) 8 AMU060 4 0.35 0.37 (CA) 16 CAUD120 7 0.71 0.76 (CA) 9 CAUD083 4 0.35 0.36 (CA) 9 AMU003 4 0.54 0.55 (CA) 7 CAUD130 4 0.7 0.51 (AC) 11 Average 4.5 0.54 0.50

As shown in Table 3, an average of 4.5 alleles appeared for each marker, and the average Obs-Ht and PIC values were 0.54 and 0.50, respectively.

The results of the allelic genotyping analysis for the 12 loci of supersatellites are shown in FIG.

As shown in FIG. 1, a specific reaction occurred in a total of 12 loci of the supersatellites, and it was confirmed that all the alleles were identified to identify individuals.

<110> GenDocs, Inc. <120> Method for identifying duck using multiplex PCR <160> 24 <170> Kopatentin 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CAUD004 forward <400> 1 tccacttggt agaccttgag 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CAUD004 reverse <400> 2 tgggattcag tgagaagcct 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CAUD055 forward <400> 3 gtttgtttgc cctgtgcttg 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CAUD055 reverse <400> 4 ctagtctggg attatgtctg a 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CAUD065 forward <400> 5 tgcaagccct ttttatcctg 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CAUD065 reverse <400> 6 gcatctaggt cagagcgttg t 21 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CAUD074 forward <400> 7 caagccctgg acctattcag 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CAUD074 reverse <400> 8 gagatgggaa gggagagagg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CAUD083 forward <400> 9 gctgcctgga atataaccgc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CAUD083 reverse <400> 10 gcatttgcag agtcaggacc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CAUD086 forward <400> 11 aacacagctt caccccacag 20 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CAUD086 reverse <400> 12 gcagagcggt gtgagagca 19 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CAUD120 forward <400> 13 aatatcctgt cgccgtggt 19 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CAUD120 reverse <400> 14 aattcttgct gagattatag ag 22 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CAUD130 forward <400> 15 ctcagtggca taaatcaggc 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CAUD130 reverse <400> 16 atatcatggg cttgttacgg 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AMU003 forward <400> 17 ttacaagccc tgcacccgga 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AMU003 reverse <400> 18 tcccagctcc cttcttggca 20 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> AMU006 forward <400> 19 tgttttctcc ctggcgttta gc 22 <210> 20 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> AMU006 reverse <400> 20 tgctgagatg tttgaaataa tgca 24 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> AMU060 forward <400> 21 gcgtgttgtt acaccagcag ga 22 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> AMU060 reverse <400> 22 caggcagctt caggtatgtg ca 22 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> APH21 forward <400> 23 cttaaagcaa agcgcacgtc 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> APH21 reverse <400> 24 agatgcccaa agtctgtgct 20

Claims (5)

SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, 17 and 18, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22, and SEQ ID NOs: 23 and 24, respectively.
A kit for identifying ducks comprising the composition of claim 1.
1) obtaining a nucleic acid sample from a duck subject to be identified;
2) amplifying the nucleic acid sample of step 1) by multiplex PCR using the primer pair of the first primer specific to the locus of super-satiety of the duck;
3) electrophoresing the amplified product of step 2); and
4) detecting alleles in the electrophoresis product of step 3) and determining a genotype.
The method according to claim 3, wherein in step 2), the locus of the supermatellites is CAUD055, CAUD065, CAUD086, CAUD074, CAUD004, AMU006, APH21, AMU060, CAUD120, CAUD083, AMU003 and CAUD130.

delete

Images